Regulation of pancreatic stellate cell function in vitro: biological and molecular effects of all-trans retinoic acid

Abstract

Pancreatic stellate cells (PSCs) are essentially involved in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. Profibrogenic mediators, such as ethanol metabolites and cytokines, induce a PSC activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts which includes a loss of the characteristic retinoid-containing fat droplets. Here, we have analysed how exogenous all-trans retinoic acid (ATRA) affects activation of rat PSCs induced by sustained culture. Bromodeoxyuridine-incorporation assays indicated an ATRA-dependent inhibition of DNA synthesis. In contrast, ATRA did not affect expression of α-smooth muscle actin, a protein typical for myofibroblasts. Quantification of [${}^3\text{H}$]proline incorporation revealed a diminished collagen production in ATRA-treated PSCs. Furthermore, zymography experiments showed that supernatants of ATRA-exposed PSC cultures contained higher levels of matrix metalloproteinase-9 but not of matrix metalloproteinase-2 than untreated controls. At the level of intracellular signalling, ATRA had no effect on extracellular signal-regulated kinase activation after incubation of PSCs with the mitogen platelet-derived growth factor (PDGF). In addition, PDGF-induced DNA binding of activator protein-1 (AP-1) transcription factors was not inhibited by ATRA treatment. Luciferase reporter gene assays, however, revealed an ATRA-dependent transrepression of AP-1 in PDGF-stimulated PSCs. Together, the results indicate that exogenous ATRA displays inhibitory effects on PSC proliferation and collagen synthesis but does not block phenotypic transition towards myofibroblasts. We hypothesise that inhibition of AP-1 signalling may be involved in the mediation of biological effects of ATRA on PSCs.

Introduction

In the development of pancreatic fibrosis, a characteristic feature of chronic pancreatitis and pancreatic cancer [1], [2], PSCs play a crucial role [3], [4], [5]. Profibrogenic mediators, such as oxidative stress, ethanol metabolites and cytokines, stimulate fibrogenesis through the induction of PSC activation; a process that involves enhanced cell proliferation, a phenotypic transition towards myofibroblasts as well as production of increased amounts of collagens (types I and III) and other extracellular matrix (ECM) proteins [3], [4], [5], [6], [7], [8]. Cytokines promoting PSC activation include the mitogen PDGF and the autocrine stimulator of ECM synthesis transforming growth factor-β (TGF-β) [4], [9], [10]. In a previous study, we have shown that at the intracellular level extracellular signal-regulated kinases (ERKs) are essentially involved in the transduction of mitogenic signals [11]. The precise molecular mechanisms which underlie the activation process, however, remain to be deciphered.

Like their hepatic counterparts, the hepatic stellate cells (HSCs), PSCs store retinoids in fat droplets [3], [6], [12]. Activation and phenotypic transition of HSCs and PSCs towards myofibroblasts correlate with a strong decrease of cellular Vitamin A reserves [3], [6], [13]. Retinoids are well-established regulators of proliferation and differentiation of various types of cells [14], raising the question whether Vitamin A depletion may be not just a phenomenon that accompanies activation of stellate cells but plays a direct role in this process. Indeed, studies with cultured HSCs have indicated inhibitory effects of exogenous biological active Vitamin A metabolites, such as retinoic acid (RA) derivatives, on cell proliferation [15], [16] and collagen type I synthesis [17], [18]. Still, the precise biological effects of the various retinoids on HSCs are incompletely known. For example, conflicting data have been reported regarding the influence of the most studied RA derivative, ATRA, on HSC growth [15], [16], [18]. More studies are also required to test the hypothesis that HSC activation could be associated with diminished RA responsiveness and signalling [19]. The biological functions and molecular effects of retinoids in pancreatic stellate cells are largely unknown.

Here, we show that the Vitamin A metabolite ATRA reduced the growth rate of stellate cells isolated from rat pancreas as well as collagen production but did not inhibit induction of α-smooth muscle actin (α-SMA) expression in the course of primary culture. Furthermore, ATRA stimulated expression of matrix metalloproteinase (MMP)-9. At the level of signal transduction, ATRA displayed characteristics of a transrepressor of AP-1, a transcription factor family (composed of Fos and Jun proteins) with complex functions in the regulation of cell growth, differentiation and survival [20]. In contrast, AP-1 DNA binding activity and ERK 1/2 activation were not affected by ATRA.